Aims

Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages.

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To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis.

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The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis.

Bone tissue is known to possess an intrinsic regeneration potential. However, in cases of major injury, trauma, and disease, bone loss is present, and the regeneration potential of the tissue is often impaired. The process of bone regeneration relies on a complex interaction of molecules. MicroRNAs (miRNA) are small, non-coding RNAs that inhibit messenger RNAs (mRNA). One miRNA can inhibit several mRNAs and one mRNA can be inhibited by several miRNAs. Functionally, miRNAs regulate the entire proteome via the local inhibition of translation. In fact, miRNA modulation has been shown to be involved in several musculoskeletal diseases. 1. In those pathologies, they modulate the transcriptional activity of mRNAs important for differentiation, tissue-specific activity, extracellular matrix production, etc. Because of their function in inhibiting translation, miRNAs are being researched in many diseases and are already being used for interventional treatment. 2. Bone tissue and its related conditions have been widely investigated up to this day. 1,3. This talk will focus on the relevancy of miRNAs to bone tissue, its homeostasis, and disease. After, examples will be given of how miRNAs can be used in bone regeneration and diseases such as osteoporosis and osteosarcoma. The use of miRNAs in both, detection and therapy will be discussed

Objectives. The aim of this study was to provide a comprehensive understanding of alterations in messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) in cartilage affected by osteoarthritis (OA). Methods. The expression profiles of mRNAs, lncRNAs, and circRNAs in OA cartilage were assessed using whole-transcriptome sequencing. Bioinformatics analyses included prediction and reannotation of novel lncRNAs and circRNAs, their classification, and their placement into subgroups. Gene ontology and pathway analysis were performed to identify differentially expressed genes (DEGs), differentially expressed lncRNAs (DELs), and differentially expressed circRNAs (DECs). We focused on the overlap of DEGs and targets of DELs previously identified in seven high-throughput studies. The top ten DELs were verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in articular chondrocytes, both in vitro and in vivo. Results. In total, 739 mRNAs, 1152 lncRNAs, and 42 circRNAs were found to be differentially expressed in OA cartilage tissue. Among these, we identified 18 overlapping DEGs and targets of DELs, and the top ten DELs were screened by expression profile analysis as candidate OA-related genes. WISP2, ATF3, and CHI3L1 were significantly increased in both normal versus OA tissues and normal versus interleukin (IL)-1β-induced OA-like cell models, while ADAM12, PRELP, and ASPN were shown to be significantly decreased. Among the identified DELs, we observed higher expression of ENST00000453554 and MSTRG.99593.3, and lower expression of MSTRG.44186.2 and NONHSAT186094.1 in normal versus OA cells and tissues. Conclusion. This study revealed expression patterns of coding and noncoding RNAs in OA cartilage, which added sets of genes and noncoding RNAs to the list of candidate diagnostic biomarkers and therapeutic agents for OA patients. Cite this article: H. Li, H. H. Yang, Z. G. Sun, H. B. Tang, J. K. Min. Whole-transcriptome sequencing of knee joint cartilage from osteoarthritis patients. Bone Joint Res 2019;8:290–303. DOI: 10.1302/2046-3758.87.BJR-2018-0297.R1

Approximately 30% of general practice consultations for musculoskeletal pain are related to tendon disorders, causing substantial personal suffering and enormous related healthcare costs. Treatments are often prone to long rehabilitation times, incomplete functional recovery, and secondary complications following surgical repair. Overall, due to their hypocellular and hypovascular nature, the regenerative capacity of tendons is very poor and intrinsically a disorganized scar tissue with inferior biomechanical properties forms after injury. Therefore, advanced therapeutic modalities need to be developed to enable functional tissue regeneration within a degenerative environment, moving beyond pure mechanical repair and overcoming the natural biological limits of tendon healing. Our recent studies have focused on developing biologically augmented treatment strategies for tendon injuries, aiming at restoring a physiological microenvironment and boosting endogenous tissue repair. Along these lines, we have demonstrated that the local application of mesenchymal stromal cell-derived small extracellular vesicles (sEVs) has the potential to improve rotator cuff tendon repair by modulating local inflammation and reduce fibrotic scarring. In another approach, we investigated if the local delivery of the tendon ECM protein SPARC, which we previously demonstrated to be essential for tendon maturation and tissue homeostasis, has the potential to enhance tendon healing. Finally, I will present results demonstrating the utility of nanoparticle-delivered, chemically modified mRNAs (cmRNA) to improve tendon repair

Aims. This study aimed to uncover the hub long non-coding RNAs (lncRNAs) differentially expressed in osteoarthritis (OA) cartilage using an integrated analysis of the competing endogenous RNA (ceRNA) network and co-expression network. Methods. Expression profiles data of ten OA and ten normal tissues of human knee cartilage were obtained from the Gene Expression Omnibus (GEO) database (GSE114007). The differentially expressed messenger RNAs (DEmRNAs) and lncRNAs (DElncRNAs) were identified using the edgeR package. We integrated human microRNA (miRNA)-lncRNA/mRNA interactions with DElncRNA/DEmRNA expression profiles to construct a ceRNA network. Likewise, lncRNA and mRNA expression profiles were used to build a co-expression network with the WGCNA package. Potential hub lncRNAs were identified based on an integrated analysis of the ceRNA network and co-expression network. StarBase and Multi Experiment Matrix databases were used to verify the lncRNAs. Results. We detected 1,212 DEmRNAs and 49 DElncRNAs in OA and normal knee cartilage. A total of 75 dysregulated lncRNA-miRNA interactions and 711 dysregulated miRNA-mRNA interactions were obtained in the ceRNA network, including ten DElncRNAs, 69 miRNAs, and 72 DEmRNAs. Similarly, 1,330 dysregulated lncRNA-mRNA interactions were used to construct the co-expression network, which included ten lncRNAs and 407 mRNAs. We finally identified seven hub lncRNAs, named MIR210HG, HCP5, LINC00313, LINC00654, LINC00839, TBC1D3P1-DHX40P1, and ISM1-AS1. Subsequent enrichment analysis elucidated that these lncRNAs regulated extracellular matrix organization and enriched in osteoclast differentiation, the FoxO signalling pathway, and the tumour necrosis factor (TNF) signalling pathway in the development of OA. Conclusion. The integrated analysis of the ceRNA network and co-expression network identified seven hub lncRNAs associated with OA. These lncRNAs may regulate extracellular matrix changes and chondrocyte homeostasis in OA progress. Cite this article:Bone Joint Res. 2020;9(3):90–98

We collected 16 samples of the membrane which surrounds loose hip prostheses from patients undergoing revision operations for aseptic loosening. To serve as the control group, samples of the synovial tissue and the fibrous capsular tissue were collected from 11 patients undergoing primary hip arthroplasties. Analyses of the expression levels of inducible nitric oxide synthase (iNOS), tumour necrosis factor-α (TNF-α), and cytosolic phospholipase A. 2. (cPLA. 2. ) mRNAs were performed by a reverse transcription polymerase chain reaction, and the content of nitrite was measured by the Griess reaction using sodium nitrite as the standard. The expression levels of iNOS, TNF-α, and cPLA. 2. mRNAs in the membranes were significantly higher than those in the control samples (p < 0.05). The expression levels of iNOS mRNA and the nitrite content in the membranes significantly correlated with those of TNF-α and cPLA. 2. mRNAs, respectively. In addition, the expression levels of iNOS, TNF-α, and cPLA. 2. mRNAs were significantly higher in membranes from cementless than in those from cemented implants (p < 0.05). Our results suggest that the expression levels of iNOS, TNF-α, and cPLA. 2. mRNAs in the membranes are regulated by closely-related mechanisms and that these have a significant role in aseptic loosening

Introduction. Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily of growth factors and are known to regulate proliferation and expression of the differentiated phenotype of chondrocytes, osteoblasts, and osteoclasts. To investigate the osteoblastic differentiation gene expressions that contribute to BMP-7 dependent ostogenesis, we performed gene expression profiling of BMP-7-treated mouse bone marrow stromal cells. Methods. D1 cells (mouse bone marrow stromal cells) were cultured in osteogenic differentiation medium (ODM) for 3 days, and then treated with BMP-7 for 24 hr. Total RNA was extracted using Trizol, purified using RNeasy columns. Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit to yield biotinylated cRNA. The data analysis up- and down-regulation developmental processes (anterior/posterior patterning, ectoderm development, embryogenesis, gametogenesis, mesoderm development, other development process, and segment specification) genes expression fold. Results. We detected 18 mRNAs (Id2, Igf2, Pparg, S100a10, Foxn3, Tulp3, Mycbp2, Notch3, Ptk7, Lrp4, Tnfrsf11b, Ogn, Cyr61, Mglap, Akp2, Ltbp4, Ibsp, and Thbs1) that were differentially up-regulated after BMP-7 stimulation. 3 mRNAs (Wars, Adss and Trim35) were differentially down-regulated after BMP-7 stimulation. Conclusion. The data indicate that BMP-7 regulate various developmental processes genes expression during osteoblastic differentiation. Though further studies are needed in relation to each expression gene profiles and osteoblastic differentiation, this information may serve as a point of comparison for osteoblastic differentiation of BMP-7. Furthermore, the data should facilitate the informed use of BMP-7 as a therapeutic agent and tissue engineering tool. Acknowledgement. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2008-000-10089-0)

Chondrocyte dysfunction is attributable to the development of osteoarthritis (OA). Deregulation of chondrogenic regulators and deleterious factors, e.g. proteinases, Wnt signalling components, and autophagy repressors lowers chondrogenic activities and ultimately deteriorates cartilage homeostasis. Emerging evidence is that epigenetic pathways, including non-coding microRNAs and histone remodelling switch on/off the expression of joint-deleterious factors. MicroRNAs reduces the expressions of mRNAs through binding to the 3'-untranslation regions of targets. The levels of microRNAs, e.g. miR-29a, miR-128a in serum, synovial fluid, synovium, and cartilage are correlated with the occurrence of OA. Mice overexpressing/deficient microRNAs of interest show minor responses to OA progression. Besides, acetylation and methylation statuses of histones regulate the factors detrimental to chondrocytes through altering the interactions between histones and promoters. Histone deacetylases and demethylases, e.g. HDAC4, SIRT1, and EZH2 contribute to the modification reactions of histones, which modulate cartilage matrix metabolism. An intricate nature is that reciprocal actions between microRNAs and histone deacetylase/demethylase are indispensable in chondrocyte survival and function. Administrations with specific inhibitor/agonists for microRNAs and histone deacetylases/demethylase enable joints to show minor responses to articular injury, which mitigate the pathogenesis of OA. This talk highlights the biological roles and therapeutic advantage of epigenetic microRNAs and histone remodelling in OA

Objectives. Osteoporosis is a metabolic disease resulting in progressive loss of bone mass as measured by bone mineral density (BMD). Physical exercise has a positive effect on increasing or maintaining BMD in postmenopausal women. The contribution of exercise to the regulation of osteogenesis in osteoblasts remains unclear. We therefore investigated the effect of exercise on osteoblasts in ovariectomized mice. Methods. We compared the activity of differentially expressed genes of osteoblasts in ovariectomized mice that undertook exercise (OVX+T) with those that did not (OVX), using microarray and bioinformatics. Results. Many inflammatory pathways were significantly downregulated in the osteoblasts after exercise. Meanwhile, IBSP and SLc13A5 gene expressions were upregulated in the OVX+T group. Furthermore, in in vitro assay, IBSP and SLc13A5 mRNAs were also upregulated during the osteogenic differentiation of MC3T3-E1 and 7F2 cells. Conclusion. These findings suggest that exercise may not only reduce the inflammatory environment in ovariectomized mice, indirectly suppressing the overactivated osteoclasts, but may also directly activate osteogenesis-related genes in osteoblasts. Exercise may thus prevent the bone loss caused by oestrogen deficiency through mediating the imbalance between the bone resorptive activity of osteoclasts and the bone formation activity of osteoblasts. Cite this article: W-B. Hsu, W-H. Hsu, J-S. Hung, W-J. Shen, R. W-W. Hsu. Transcriptome analysis of osteoblasts in an ovariectomized mouse model in response to physical exercise. Bone Joint Res 2018;7:601–608. DOI: 10.1302/2046-3758.711.BJR-2018-0075.R2

Autologous chondrocyte transplantation is a widely used technique for the treatment of cartilage lesions. This therapeutic strategy has been recently improved by the use of biocompatible scaffolds which allow a better fixation of the cells inside the defect together with the maintenance of their original phenotype. We have recently reported that human chondrocytes can efficiently grow on a hyaluronan acid derivative biomaterial (Hyaff-11, Fidia Advanced Biopolymers, Abano Terme, Italy) and are able to express and produce collagen type II and proteoglycans, molecules expressed by differentiated cells (Grigolo et al. Biomaterials 2002). However, from the histological evaluations of the grafted tissues there is not always evidence of hyaline cartilage neo-formation even in presence of good clinical symptoms. Only few studies deals with cellular, and biochemical processes that occur during the remodeling of the graft tissue after transplantation in humans. Biopsy samples harvested from the graft have been examined using a panel of specific antibodies. It was found that cell transplantation is followed not only by a process of cartilage repair but in some cases also by a regeneration achieved through the turnover of the initial fibrocartilagineous tissue via enzymatic degradation and synthesis of newly formed collagen type II. Therefore, we examined the expression of genes encoding extracellular matrix proteins and regulatory factors essential for cell differentiation in human cartilage biopsies of patients who underwent autologous chondrocyte transplantation. Human cartilage biopsies of patients treated by autologous chondrocyte transplantation and from a multi-organ donor were used. A Real-Time RT-PCR analysis was performed in isolated chondrocytes to evaluate the expression of collagen type I, II, X, aggrecan, cathepsin B, early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for ECM proteins and regulatory proteins was carried out on paraffin embedded sections. Real-time RT-PCR analysis showed that collagen type I mRNA was expressed in all the samples evaluated while collagen type II was present even if at lower levels compared to control. Collagen type X messenger was undetectable. Aggrecan mRNA was present in all the samples at lower levels compared to donor. Cathepsin B messenger was higher in the samples compared to control. Egr-1 and Sox-9 mRNAs were expressed at lower levels compared to donor. The immunohistochemical analysis showed a slight positivity for collagen type I in all the sections. Collagen type II was found in all the samples evaluated with a positivity confined inside the cells, while the control displayed a positivity which was diffuse in the ECM. Cathepsin B was slightly positive in all the samples while the control was negative. Egr-1 protein was particularly evident in the areas negative for collagen type II. Sox-9 was positive in all the samples, with evident localization in the superficial layer. Our results provide evidence that the remodelling of the graft tissue after autologous chondrocyte transplantation is regulated by a sophisticated gene expression machinery control addressed to new cartilage formation

Multiple myeloma (MM) is an incurable hematological tumor stemming from malignant plasma cells. MM cells accumulate in the bone marrow (BM) and shape the BM niche by establishing complex interactions with normal BM cells, boosting osteoclasts (OCLs) differentiation and causing bone disease. This unbalance in bone resorption promotes tumor survival and the development of drug resistance. The communication between tumor cells and stromal cells may be mediated by: 1) direct cell-cell contact; 2) secretion of soluble factors, i.e. chemokines and growth factors; 3) release of extracellular vesicles/exosomes (EVs) which are able to deliver mRNAs, miRNAs, proteins and metabolites in different body district. Primary CD138+ MM cells were isolated from patients BM aspirates. MM cell lines were cultured alone in complete RPMI-1640 medium or co-cultured with murine (NIH3T3) or human (HS5) BMSC cell lines or murine Raw264.7 monocytes in DMEM medium supplemented with 10% V/V FBS. Silencing of Jagged1 and Jagged2 was obtained by transient expression of specific siRNAs or by lentiviral transduction using a Dox-inducible system (pTRIPZ). EVs were isolated using differential ultracentrifugation. EVs concentration and size were analyzed using Nano Track Analysis (NTA) system. The uptake of PKH26-labelled MM-derived EVs by HS5 or Raw264.7 was measured after 48 hours by confocal microscopy and flow cytometry. Osteoclast (OCL) differentiation of Raw264.7 cells was induced by 50ng/ml mRANKL, co-culturing with MM cells, CM or EVs. OCLs were stained by TRAP Kit and counted. Bone resorption was assessed by Osteo Assay Surface plates. Flow cytometric detection of apoptotic cells was performed after staining with Annexin V. Gene expression was analyzed by qRT-PCR, while protein levels were determined using flow cytometry ELISA or WB. Notch oncogenic signaling is dysregulated in several hematological and solid malignancies. Notch receptors and ligands are key players in the crosstalk between tumor cells and BM cells. We have demonstrated that: 1) the dysregulated Jagged ligands on MM cells trigger the activation of Notch receptors in the nearby stromal cells by cell-cell contact. This results in the release of anti-apoptotic and growth stimulating factors, i.e. IL6 and SDF1; 2) MM cells promote the development of bone lesions boosting osteoclast differentiation by secreting soluble factors (i.e. RANKL) and by the activation of Notch signaling mediated by direct contact with osteoclast precursors; 3) Finally, we present evidences that EVs play a crucial role in the dysregulated interactions of MM cells with the microenvironment and that Notch signaling regulates their release and participate in this cross-talk. These evidences supports the hypothesis that Jagged targeting on MM cells may interrupt the communication between tumor cells and the surrounding milieu, blocking the activation of the oncogenic Notch pathway and finally resulting in the a reduction of MM-associated bone disease and drug resistance

Aims

Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP.

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In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD.

Periprosthetic bone loss after total joint arthroplasty is a major clinical problem resulting in aseptic loosening of the implant. Among many cell types, osteoblasts play a crucial role in the development of peri-implant osteolysis. In this study, we tested the effects of calcitriol (1α,25-dihydroxy-vitamin-D. 3. ) and the bisphosphonate pamidronate on titanium-particle- and TNF-α-induced release of interleukin-6 and suppression of osteoblast-specific gene expressions in bone-marrow-derived stromal cells with an osteoblastic phenotype. We monitored the expression of procollagen α1[1], osteocalcin, osteonectin and alkaline phosphatase mRNAs by Northern blots and real-time reverse transcription and polymerase chain reaction analyses. The release of various cytokines was also analysed by ELISA. We found that calcitriol or pamidronate could only partially recover the altered functions of osteoblasts when added alone. Only a combination of these compounds restored all the tested functions of osteoblasts. The local delivery of these drugs may have therapeutic potential to prevent or to treat periprosthetic osteolysis and aseptic loosening of implants

A variety of scaffolds, including collagen-based membranes, fleeces and gels are seeded with osteoblasts and applied for the regeneration of bone defects. However, different materials yield different outcomes, despite the fact that they are generated from the same matrix protein, i.e. type I collagen. Recently we showed that in fibroblasts MMP-3 is induced upon attachment to matrix proteins in the presence of TGFbeta. Aim: To investigate the regulation of matrix metalloproteinases (MMPs) and interleukins (IL) in osteoblasts upon attachment to type I collagen (col-1) in comparison to laminin -1 (LM-111) in the presence or absence of costimulatory signals provided by transforming growth factor beta (TGFbeta). Methods: Osteoblasts were seeded in col-1–and LM-111-coated flasks and activated by the addition of TGFbeta. Mock-treated cells served as controls. The expression of genes was investigated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunocytochemistry and ELISA. Results: Attachment of osteoblasts to col-1 or LM-111 failed to activate the expression of MMPs or ILs. In contrast, TGFbeta induced the expression of MMP-3, MMP-9, and MMP-13, IL-6 and IL-16 mRNAs. MMP-3 was found to be elevated in supernatants of activated cells. No difference was found in the expression of MMP-1, IL-8 and IL–18. Interestingly, the expression of IL-1beta mRNA was not activated by TGFbeta alone, but it was activated by attachment of osteoblasts to LM-111 in the presence of TGFbeta. Conclusion: In contrast to fibroblasts, attachment of osteoblasts to col-1 or LM-111 had no effect on the induction of MMPs and ILs. TGFbeta induced the expression of MMPs and ILs in these cells but only MMP-3 was released. The results show significant differences between osteoblasts and fibroblasts in the effects of attachment to scaffold materials. This may have important consequences for tissue engineering of bone and for wound healing after surgery

Mechanical loading is a potent stimulator of bone formation. A screen for genes associated with mechanically-induced osteogenesis implicated the glutamate transporter GLAST-1 (1), in the mechanoresponse. We are investigating whether modulation of glutamate transporters represents a potential anabolic therapy in bone. Bone cells express functional components from each stage of the glutamate signalling pathway and activation of ionotropic glutamate receptors on osteoblasts can increase bone forming activity (2). Five high affinity Na+-dependant excitatory amino acid transporters (EAATs 1-5) regulate glutamatergic signalling. EAAT1 (GLAST-1) is expressed by osteocytes and bone-forming osteoblasts in vivo. We quantified transcripts for EAATs 1-3 and two splice variants (EAAT1a and EAAT1ex9skip) in human osteoblasts (MG63, SaOS-2 and primary) using real time-PCR. EAAT1a expression was very low whilst levels of the dominant negative EAAT1ex9skip were much higher in all cell types. EAAT1 and EAAT3 proteins were detected by immunofluorescence. We also demonstrated that glutamate transporters function in human osteoblasts. Sodium-dependent 14C-labelled glutamate uptake, sensitive to pharmacological EAAT inhibitors (t-PDC, TBOA) and extracellular glutamate concentration (10-500μM) was detected in MG63 and SaOS-2 cells. To determine whether modulation of EAATs can influence bone formation, we used pharmacological inhibitors of EAATs 1-5 (t-PDC and TBOA) and also over-expressed EAAT1exon9skip using antisense oligonucleotides (AONs) targeted to splice donor sequence of exon 9. Experiments were performed in 0-500μM glutamate. Pharmacological inhibition of EAATs over 5-21 days increased alkaline phosphatase activity and mineralisation of SaOS-2 cells and human primary osteoblasts. Over-expression of EAAT1ex9skip significantly increased cell number and decreased cell death as well as significantly increasing PCNA, Osteonectin and Type I collagen mRNAs in MG63 cells. Furthermore, over-expression of EAAT1ex9skip increased mean alkaline phosphatase activity over 48hrs in SaOS-2 cells. These data show that EAATs are expressed and functional in osteoblasts and that pharmaceutical and genetic inhibition of their activity increases bone formation. These mechanically regulated glutamate transporters are important in regulating bone homeostasis and their manipulation may represent a new anabolic therapy for the treatment of disorders such as osteoporosis or non-union fractures

Purpose of the study: Since glutamate can activate both nociceptive and pathological processes, we have investigated glutamate signalling in patients with painful and asymptomatic meniscal tears to determine which components are expressed, whether this varies in different anatomical regions of the meniscus and whether it is influenced by pain or degeneration. Methods and results: Meniscus samples were obtained from two patients undergoing arthroscopic partial meniscal resection for chronic degenerate painful meniscal tears, from one patient with a torn painless meniscus and from the less affected compartment of the knee joint of three patients undergoing total knee arthroplasty. Menisci were dissected into anatomical regions (anterior horn, body, posterior horn, inner vascular, outer avascular), cryosectioned and RNA extracted. RNA was reverse transcribed and PCR performed for the housekeeping gene GAPDH and glutamate receptor subunits (NR2A, AMPA GluR3, KA1). Absolute quantitative RT-PCR assessed mRNA expression of glutamate transporters (EAAT-1, EAAT-1ex9skip) and type I collagen after normalisation to GAPDH or total RNA. Human meniscus expressed GAPDH, type 1 collagen, EAAT-1, EAAT-1ex9skip, NR2A, AMPA GluR3 and KA1 mRNAs. Levels of EAAT-1 expression, normalised to GAPDH, did not differ between the inner and outer halves, or in the anterior, middle or posterior regions of menisci from the less affected compartments of arthritic knees. EAAT-1 expression appeared greater in the 2 painful, compared with the single non-painful meniscus. Interestingly, EAAT-1ex9skip was significantly more common within the outer zones (ANOVA, P=0.040) and in the posterior horns of the menisci (ANOVA, p=0.038). Conclusion: We have shown for the first time that glutamate receptors and transporters are expressed in human meniscus providing a potential mechanism underlying the pathophysiology of pain associated with a torn meniscus. Our preliminary data indicate that EAAT-1 and EAAT-1ex9skip expression may vary with extent of damage and anatomical location in the human meniscus

Background: In regenerative medicine the autologous cartilage implantation (ACI) has been used for the repair of cartilage defects. As modification of ACI, the matrix assisted ACI is used nowadays with varying results. There is a general discussion about whether supporting scaffolds should be used or whether a scaffold-free cartilage repair is the method of choice. The major problem of scaffold-free regenerates is how to keep the cells in place after transplantation. Aim of this study was to examine a new scaffold-free diffusion-culture model, which uses a mega-congregate of chondrocytes cultured at an air-medium interface. This scaffold-free high-density diffusion culture could be used to repair cartilage defects. Material and methods: Human chondrocytes from passage 1–7 were expanded in monolayer and transferred to pellet-culture or diffusion-culture. After one week cultures were stained with toluidine blue and safranin-O and evaluated by immunohistochemical staining for type II collagen. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for the mRNAs of cartilage markers. Results: Positive alcian blue staining was detectable in diffusion-culture for human chondrocytes up to passage 7. Within passages the amount of proteoglycan production in relationship to the number of cells increased. There was a positive signal for Collagen type II in diffusion-cultures up to passage 7. In qRT-PCR a redifferentiation of human chondrocytes was shown by the transfer into diffusion-culture. Within passage 1 to 3 human chondrocytes which were cultured in monolayer lost the ability to express Collagen Type II but could regain it if they were transferred to diffusion-culture. At diffusion-culture chondrocytes showed the highest expression of Collagen type II at passage 1 when compared to monolayer or to pellet-culture. Conclusion: It could be shown that the cultivation in a scaffold-free diffusion-culture can lead to redifferentiation of human chondrocytes Chondrocytes in diffusion-cultures tend to form their own matrix and produce Collagen type II at higher amounts than in monolayer or in normal pellet-cultures. Therefore diffusion-culture congregates might be an appropriate tool to be used for a new scaffold-free cartilage regeneration approach